Abstract
Once two radioactive Ca2+ coming from the cytoplasm are bound to the transport sites of the nonphosphorylated ATPase, excess EGTA induces rapid dissociation of both ions, whereas excess nonradioactive Ca2+ only reaches one of the two bound Ca2+. This difference has been explained assuming that the two Ca2+ sites are in a single file channel in which the superficial Ca2+ is freely exchangeable from the cytoplasm, whereas the deeper Ca2+ is exchangeable only when the superficial site is vacant. The same experiment was done using phosphorylated ATPase to determine whether Ca2+ dissociation toward the lumen is sequential as well. Under conditions that allow ADP-sensitive phosphoenzyme to accumulate (leaky vesicles, 5 degrees C, pH 8, 300 mM KC1), we found the same two pools of Ca2+. Excess EGTA induced dissociation of both ions together with dephosphorylation. Excess nonradioactive Ca2+ induced the exchange of half the radioactive Ca2+ without any effect on the phosphoenzyme level. Our results show a close similarity between the transport sites of the nonphosphorylated and the phosphorylated enzymes, although the orientation, affinities, and dissociation rate constants are different.
Highlights
From the Commissariat a l'Energie Atomique and Unite de Recherche 1290 Assoezee au Centre National de la Recherche
Once two radioactive Ca2 + coming from the cytoplasm are bound to the transport sites of the nonphosphorylated ATPase, excess EGTA induces rapid dissociation of both ions, whereas excess nonradioactive Ca2 + only reaches one of the two bound Ca2 +
The transport sites change their orientation and affinity, depending on whether the ATPase is phosphorylated, The high-affinity transport sites of the nonphosphorylated ATPase are accessible from the cytoplasm, whereas once the ATPase has been phosphorylated the transport sites have lower affinity and are accessible from the lumen
Summary
From the Commissariat a l'Energie Atomique and Unite de Recherche 1290 Assoezee au Centre National de la Recherche. Once two radioactive Ca2 + coming from the cytoplasm are bound to the transport sites of the nonphosphorylated ATPase, excess EGTA induces rapid dissociation of both ions, whereas excess nonradioactive Ca2 + only reaches one of the two bound Ca2 +. The transport sites change their orientation and affinity, depending on whether the ATPase is phosphorylated, The high-affinity transport sites of the nonphosphorylated ATPase are accessible from the cytoplasm, whereas once the ATPase has been phosphorylated the transport sites have lower affinity and are accessible from the lumen This allows Ca 2+ release into the SRI lumen and is followed by dephosphorylation of the phosphoenzyme. By monitoring the internalization of the Ca 2+ ions after phosphorylation, Inesi [5] concluded that their dissociation toward the lumen is sequential and that the first Ca2+ bound to E is the first to be internalized
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
